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Involvement of 5-HT2A Receptor in the Regulation ofHippocampal-Dependent Learning and Neurogenesis

by

Briony J Catlow

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of PhilosophyDepartment of Psychology College of Arts and Sciences University of South Florida

Date of Approval: November 7, 2008

© 2008, Briony J Catlow

original source: http://digital.lib.usf.edu/SFS0027045/00001/pdf

Major Professor: Cheryl Kirstein, Ph.D.Michael Brannick, Ph.D.Cindy Cimino, Ph.D.Juan Sanchez-Ramos, M.D.Toru Shimizu, Ph.D.

Keywords: neurogenesis, serotonin, hippocampus, fear conditioning, psilocybin

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Table of Contents:AcknowledgementsAbstractChapter One Introduction The Anatomy of Hippocampal Neurogenesis Regulation of Neurogenesis in the Dentate Gyrus Hippocampal Neurogenesis and Learning Assessing Neurogenesis Serotonergic Innervation in the Dentate Gyrus Serotonin and Neurogenesis in the Dentate Gyrus Psilocybin (PSOP) Specific Aims Specific Aim 1 Specific Aim 2Chapter Two - Involvement of the 5HT2A receptor in the Regulation of Adult Neurogenesis in theMouse Hippocampus Abstract Introduction Materials and Methods Subjects Drugs General Procedure Immunofluorescence Quantitation

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Design and analyses Results Effects of Acute Administration of 5-HT2A receptor agonists and an antagonist in vivo on Cell Survival andNeurogenesis in the Hippocampus Effects of Repeated Intermittent PSOP Administration on Progenitor Cell Survival and Neurogenesis in theHippocampus Discussion AcknowledgmentsChapter Three - The Effects of Psilocybin on Trace Fear Conditioning Abstract Introduction Materials and Methods Subjects General Procedure Design and analyses Results Acquisition Contextual Fear Conditioning Cue Fear Conditioning Discussion AcknowledgmentsReference List

AcknowledgementsFirst and foremost thank you to Dr Kirstein and Dr Sanchez-Ramos for their complete support during mygraduate studies. Dr Kirstein, you fought for me from the beginning and I am so grateful because I know noneof this would have been possible without your advice and support. Dr Sanchez, you have taught me to thinkabout the brain in a holistic way and with your passion for knowledge I learned that neuroscience is more thanjust a career, it is a way of life. Dr Paula Bickford, you are one of the most generous people I know, thank youfor support and guidance and blessing me in so many ways. To Dr Brannick, Dr Cimino and Dr Shimizu, thankyou for taking the time to serve on my committee and for your thoughtful consideration of my projects. I wouldalso like to thank Dr Naomi Yavneh for chairing my defense.

On a personal level I am so grateful to have had the support of my family. To my Grandma, Nana, Mum, Dad,Pam, Steve, Jodi and Joanie you have all supported me in ways only family can and hope that I can do the samefor you. To Anne, you have always been a role model to me with beauty, smarts, and positivity and I am solucky to have you in my life. To Danielito, you opened my eyes and held my hand, ahora vamos!

AbstractAberrations in brain serotonin (5-HT) neurotransmission have been implicated in psychiatric disordersincluding anxiety, depression and deficits in learning and memory. Many of these disorders are treated withdrugs which promote the availability of 5-HT in the synapse. Selective serotonin uptake inhibitors (SSRIs) areknown to stimulate the production of new neurons in the hippocampus (HPC) by increasing synapticconcentration of serotonin (5-HT). However, it is not clear which of the 5-HT receptors are involved inbehavioral improvements and enhanced neurogenesis. The current study aimed to investigate the effects of5HT2A agonists psilocybin and 251-NBMeO and the 5HT2A/C antagonist ketanserin on neurogenesis andhippocampal-dependent learning. Agonists and an antagonist to the 5-HT2A receptor produced alterations inhippocampal neurogenesis and trace fear conditioning. Future studies should examine the temporal effects ofacute and chronic psilocybin administration on hippocampal-dependent learning and neurogenesis.



Chapter One

IntroductionThe idea of new neurons forming in the adult central nervous system (CNS) is a relatively new one. In the1960's Joseph Altman published the first evidence of neurogenesis, or the birth of new neurons in the adultmammalian brain (Altman, 1962; Altman, 1963; Altman & Das, 1965). Utilizing the tritiated thymidine method(Sidman et al., 1959; Messier et al., 1958; Messier & Leblond, 1960) Joseph Altman was able to demonstratethat the subventricular zone (SVZ) of the lateral ventricles and the dentate gyrus (DG) of the hippocampus(HPC) produce new neurons throughout the lifespan (Altman, 1962; Altman & Das, 1965; Altman, 1969). Foryears following Altman's discovery scientists acknowledged the possibility of the generation of new glial cellsin the adult brain but rejected the concept of new born neurons. With the advent of new technologies such asthe bromodeoxyuridine (BrdU) method of birth dating cells and double labeling using immunofluorescence,adult neurogenesis has been identified in many mammalian species including mice (Kempermann et al., 1998),rats (Kaplan & Hinds, 1977), hamsters (Huang et al., 1998), tree shrews (Gould et al., 1997), nonhumanprimates (Gould et al., 1999b; Bernier et al., 2002) and humans (Eriksson et al., 1998). Peter Eriksson'sdiscovery of new neurons in the human HPC changed the perception of neurogenesis in the scientificcommunity so now the fact that new neurons are produced in the adult brain is firmly established.

The Anatomy of Hippocampal NeurogenesisThe HPC is divided into four areas: DG (also called area dentate, or fascia dentata), cornu ammonis (CA,further divided into CA1, CA2, CA3 and CA4), the presubiculum and the subiculum. This anatomicaldescription of the HPC has been confirmed by both gene expression and fiber connections. The DG and areasCA form a trisynaptic circuitry within the HPC (see Figure 1.1). Neurons in the entorhinal cortex (EC) projectto dendrites of the granule cells in the DG forming the perforant pathway. The granule cells extend their axons(Ramon, 1952) to pyramidal neurons in area CA3, forming the mossy fiber tract (Ribak et al., 1985). CA3pyramidal neurons project to the contralateral (via associational commissural pathway) and the ipsilateral CA1region forming the Shaffer collateral pathway. Pyramidal neurons in CA1 extend axons to the subiculum andfrom the subiculum back to EC (for detailed descriptions of hippocampal circuitry see (Witter, 1993)).

Figure 1.1. Anatomy of the Hippocampus. The HPC forms a trisynaptic pathway with inputs from the Entorhinal Cortex (EC) thatprojects to the Dentate Gyrus (DG) and CA3 pyramidal neurons via the perforant pathway. Granule cells in the DG project to CA3 viathe mossy fiber pathway. Pyramidal neurons in CA3 project to both the contralateral (associational commissural pathway) and theipsilateral CA1 region via the Schaffer Collateral Pathway. CA1 pyramidal neurons send their axons to the Subiculum (Sb) which inturn projects out of the HPC back to the EC.



In normal physiological conditions, neurogenesis that occurs in the HPC is found only in the DG and results inthe generation of new granule cells. Within the DG, progenitor cells reside in a narrow band between the DGand the hilus (also called CA4 or plexiform layer) called the subgranular zone (SGZ) which is approximately 2-3cells thick (20-25 µM) (see Figure 1.2). Neural progenitor cells (1) divide and form clusters of proliferatingcells (2). Proliferating cells exit from the cell cycle and begin to differentiate into immature neurons (3). Theimmature granule cell forms sodium currents, extends dendrites and an axon to make connections with othercells and form synapses to become a mature neuron (4). The SGZ contains many cell types including astrocytes(Seri et al., 2001; Filippov et al., 2003; Fukuda et al., 2003), several types of glial and neuronal progenitor cells(Filippov et al., 2003; Fukuda et al., 2003; Kronenberg et al., 2003; Seri et al., 2004) and neurons in all stagesof differentiation and maturation (Brandt et al., 2003; Ambrogini et al., 2004).

Figure 1.2. Neurogenesis in the Dentate Gyrus of the Hippocampus. Neural stem cells exist the SGZ of the DG, these cells then divide,differentiate and mature into their phenotypic fate. Neural progenitor cells (1) divide and form clusters of proliferating cells (2). Proliferatingcells exit from the cell cycle and begin to differentiate into immature neurons (3). The immature granule cell forms sodium currents,dendrites and extends an axon out to make connections with other cells and form synapses to become a mature granule cell (4).

Regulation of Neurogenesis in the Dentate GyrusThe proliferation and survival of neural progenitors in the adult HPC can be influenced in a positive andnegative manner by a variety of stimuli. Factors as diverse as stress, odors, neurotrophins, psychoactive drugssuch as antidepressants, opioids and alcohol, electroconvulsive therapy, seizures, ischemia, cranial irradiation,physical activity, learning, hormones and age amongst many others have been linked to the regulation ofneurogenesis (Kempermann et al., 1998; Van et al., 1999b; Malberg et al., 2000; Malberg & Duman, 2003;Tanapat et al., 2001). Some of these factors have been studied extensively and their role in the regulation ofneurogenesis is well defined. For example, environmental enrichment and physical activity are strong positiveregulators of neurogenesis (Van et al., 1999b; Kempermann et al., 1997), while stress and age (Cameron et al.,1993; Kempermann et al., 1998) appear to be negative regulators of neurogenesis.

The first report of any factor increasing neurogenesis in the mammalian brain was an enriched environment.In an experimental setting a rodent enriched environment typically consists of a large cage, a large number ofanimals, toys and a tunnel system. In order to maintain the enrichment aspect novel toys are introduced andtunnel system is rearranged on a regular basis. Mice (Kempermann et al., 1997) and rats (Nilsson et al., 1999)living in an enriched environment exhibited a strong up-regulation of cell proliferation and neurogenesis in theDG of the HPC. The proneurogenic effects of environmental enrichment can be increased further depending onthe age of the animal when exposure occurs. When late adolescent/young adult animals live in an enrichedenvironment it enhances the ability of environmental enrichment to up-regulate cellular proliferation andneurogenesis in the DG. In fact, when the morphology of the HPC was examined later in life, a greater numberof absolute granule cells was observed (Kempermann et al., 1997). In aged animals (typically 18 months orolder in rodents) lower levels of neurogenesis have been observed, however living in an enriched environmentcounteracts the effects of aging (Kempermann et al., 1998). Kempermann and colleagues demonstratedenvironment enrichment during aging increases cell proliferation and neurogenesis in the DG (Kempermann etal., 1998). Furthermore, if animals live in an enriched environment during mid age, basal levels ofneurogenesis increase as much as five fold in old age.

When experimentation with environmental enrichment began, novel foods were included as apart of theenvironmental enrichment experience. When similar food was given to mice living either an enriched orcontrol environment, the effect of environmental enrichment was still present. One type of diet however, has



been found to have positive effects on neurogenesis specifically, caloric restriction (Lee et al., 2000). As anexperimental manipulation caloric restriction usually consists of limiting the amount of food an animal can eatby a third. Caloric restriction is the only factor that has been shown experimentally to increase the life span ofanimals and it is thought that caloric restriction actually acts as a mild stressor. It is of interest to note whileenvironmental enrichment is a strong positive regulator of adult hippocampal neurogenesis, it does not affectadult neurogenesis in the olfactory system (Brown et al., 2003).

Exposure to an enriched environment increases neurogenesis in the DG of adult rodents, however,environmental enrichment typically includes a running wheel and increased physical activity. Physical activityis known to up-regulate cell proliferation and neurogenesis in the DG. Rodents will take full advantage of theopportunity to exercise on a running wheel during their active phase of their day. Mice have been reported torun between 3 and 8 km per night on a running wheel (Van et al., 1999a; Van et al., 1999b). Voluntary physicalactivity has been shown to increase the number of progenitor cells and new neurons in the DG of the HPC (Vanet al., 1999a; Van et al., 1999b). The effect of running on neurogenesis is acute so that running must continueto effect neurogenesis and once the animal no longer uses the running wheel the effect on neurogenesis willdecline. The up-regulation of adult neurogenesis by physical activity also increases long term potentiation(LTP) in the DG and enhances performance on the Morris water maze (MWM) (Van et al., 1999a). The MWM isa behavioral task that assesses memory and learning, but as part of the task the animals are placed into a poolof water and forced to swim. Some have argued that swimming, being physical activity, could also influencethe outcome of the task. This point was addressed by Ehninger et al. who found involuntary physical activity(swimming in radial arm water maze (RAWM)) had no effect on hippocampal neurogenesis (Ehninger &Kempermann, 2003).

The mechanisms underlying the increase in neurogenesis by physical activity are unknown however, growthfactors such as insulin growth factor -1 (IGF-1), vascular endothelial growth factor (VEGF), and brain derivedneurotrophic factor (BDNF) have been strongly implicated. IGF-1 levels are increased in the HPC of runninganimals and running induced increases in cellular proliferation and neurogenesis (Carro et al., 2001; Carro etal., 2000; Trejo et al., 2001). This increase in neurogenesis is blocked by scavenging circulating IGF-1 absentin IGF-1 mutants (Carro et al., 2000). VEGF is necessary for the effects of running on adult hippocampalneurogenesis. Blocking peripheral VEGF abolished the running-induced induction of neurogenesis, howeverthere were no detectable effects on baseline neurogenesis in non-running animal (Fabel et al., 2003).Quantitative polymerase chain reaction analysis revealed BDNF mRNA levels are significantly increased in theDG of running rats (Farmer et al., 2004). BDNF is a key factor involved in modulating neuroplasticity includingLTP and neurogenesis. Infusions of BDNF into the lateral ventricles induced neurogenesis originating in theSVZ (Pencea et al., 2001) and BDNF knockout (KO) mice have diminished levels of neurogenesis in the DG(Lee et al., 2002). Like environmental enrichment, physical activity up-regulates adult hippocampalneurogenesis, however, it does not affect adult neurogenesis in the olfactory system (Brown et al., 2003).

Stress severely impairs hippocampal neurogenesis. One of the first studies to link stress to hippocampalneurogenesis was conducted by Gould and colleagues (1992). They found stress increased the number of dyingcells in the HPC but that the total number of granule cells in the dentate was not different from non-stressedcontrols and concluded neurogenesis must be occurring to maintain cellular balance (Gould et al., 1992). Theypostulated the stress hormone, cortisol in humans and corticosterone in rodents mediates the stress effect onneurogenesis and went on to discover adrenalectomy (removing the adrenal gland hence the source ofendogenous corticosterone) led to an up-regulation of neurogenesis and exogenous corticosterone down-regulated cellular proliferation and neurogenesis in the DG (Cameron et al., 1993). Since these earlyexperiments, severe stress has been shown to downregulate cell proliferation and consecutive stages ofneuronal development using many different paradigms. Prenatal stress caused learning deficits and haddetrimental effects on neurogenesis that lasted well into adulthood (Lemaire et al., 2000). The effects ofpsychosocial stress on neurogenesis were demonstrated using the resident-intruder model of territorial treeshrews (Gould et al., 1997). Tree shrews are extremely territorial and guard their environment so theintroduction of an intruder to the resident's cage is extremely stressful. The territorial tree shrews compete fordominance and soon after the introduction of an intruder a dominant-subordinate relationship is establishedresulting in elevated cortisol and decreased neurogenesis in the subordinate tree shrew (Gould et al., 1997).Predator odor triggered a stress response in prey and had detrimental effects on cell proliferation. In rodentmodels, fox odor has been shown to decrease cell proliferation and neurogenesis in the DG (Tanapat et al.,2001). Both acute and chronic restraint stress have been shown to affect the rate of adult hippocampal



neurogenesis. Pham and colleagues demonstrated that 6 weeks of daily restraint stress suppressed cellproliferation and attenuated survival of the newly born cells, resulting in a 47% reduction of granule cellneurogenesis (Pham et al., 2003). Neurogenesis is not only affected by environmental stimuli, the absence ofstimuli, such as social isolation, negatively regulated neurogenesis. Young rats reared in social isolation for 4-8weeks showed decreased performance on the MWM and decreased hippocampal neurogenesis (Lu et al.,2003). In the learned helplessness model of depression animals are exposed to an inescapable foot shock usingavoidance testing. Exposure to inescapable shock decreased cell proliferation in the HPC, extending previousstudies demonstrating downregulation of neurogenesis by exposure to acute stressors (Malberg & Duman,2003).

The key mechanism underlying the negative impact that stress has on neuroplasticity appears to be stresshormone (glucocorticoid) secretion (Cameron et al., 1993). Acute, severe and sometimes traumatic stress leadsto chronically high levels of glucocorticoids and alters the functioning of the hypothalamic-adrenal-pituitary(HPA) axis resulting in disregulation of glucocorticoid secretion and receptor expression. Depression is anexample of a clinical condition associated with disturbed regulation of the HPA axis which upsets the circadianrhythm of hormone secretion resulting in chronically elevated glucocorticoid levels and decreasedneurogenesis (Jacobs et al., 2000).

Aging is another factor known to have a strong negative influence on neurogenesis. This has been known sincethe discovery of adult neurogenesis by Altman and Das in 1965. In the original study a progressive decrease inthe levels of neurogenesis was observed after puberty and continued into old age (Altman & Das, 1965) andthis finding has been since replicated in both rats (Seki & Arai, 1995; Kuhn et al., 1996; Cameron & McKay,1999; Bizon & Gallagher, 2003), mice (Kempermann et al., 1998) and humans (Eriksson et al., 1998). Thehighest levels of adult neurogenesis occurred in young adulthood and steadily decreased over the lifespan. Inold age (typically 18 months or older in rodents) baseline levels of neurogenesis are extremely low, however,there are ways to enhance neurogenesis in the aging hippocampus. Environment enrichment during agingincreases cell proliferation and neurogenesis in the DG, however, the effect of an enriched environment ismore robust in young animals (Kempermann et al., 1998). Animals that lived in an enriched environmentstarting at mid age had five fold increases in basal levels of neurogenesis in old age (Kempermann et al.,1998). Cortisol (or corticosterone in rodents) levels are elevated in aging which likely reduces baselineproliferation and neurogenesis. Adrenalectomy in aged animals restored adult neurogenesis in the DG to alevel comparable to that of a much younger age, demonstrating corticosterone is at least in part responsiblefor the decline in neurogenesis observed in aging (Cameron & McKay, 1999). IGF-1 levels are increased in theHPC running animals and running induced increases in cellular proliferation and neurogenesis (Carro et al.,2001; Carro et al., 2000; Trejo et al., 2001). Similarly, aged animals administered exogenous IGF-1 to restoreendogenous IGF-1 levels to that of a younger age and induced neurogenesis above controls thus counteractedthe negative effect of aging on neurogenesis (Lichtenwalner et al., 2001).

BDNF is considered a critical secreted factor modulating brain plasticity. Physical activity, which is known topositively regulate neurogenesis and induce LTP, induces hippocampal BDNF mRNA expression. It is thoughtthat BDNF may modulate the effect that physical activity has on LTP and neurogenesis (Farmer et al., 2004).Infusions of BDNF into the lateral ventricles induced neurogenesis originating in the SVZ (Pencea et al., 2001)and BDNF KO mice have diminished levels of hippocampal neurogenesis (Lee et al., 2002). In pathologicalconditions such as depression, BDNF blocks neurogenesis (which is opposite to healthy animals) and it is nowunderstood one of the critical functions of BDNF is to keep neurogenesis within a physiological range. BDNFfunction has been implicated in the neurogenesis hypothesis of depression, the idea being that theantidepressants enhance neurogenesis, and BDNF is a key regulator of this mechanism (Jacobs et al., 2000;D'Sa & Duman, 2002). Antidepressants (including selective serotonin reuptake inhibitors (SSRIs)) induce thephosphorylation of CREB, after which CREB binds to the BDNF promoter and induces BDNF transcription. 5-HT2A receptor agonists, such as 2,5-dimethoxy-4-iodoamphetamine (DOI), increase BDNF mRNA expression inthe HPC (Vaidya et al., 1997). BDNF is involved in inducing neuronal differentiation possibly through theinduction of neuronal nitric oxide synthase (nNOS) which has been shown to stop proliferation and promotedifferentiation. In vitro BDNF is a differentiation factor that can down-regulate precursor cell proliferation(Cheng et al., 2003).



Hippocampal Neurogenesis and LearningMemory involves the encoding, storing and recalling of information. The HPC plays a critical role in learningand memory by converting short-term memories into long-term memories and is pivotal in the encoding,consolidation and retrieval of episodic memory (Squire et al., 1992; Squire, 1992). Several studies haveinvestigated the connection between learning and hippocampal neurogenesis. Hippocampal mediated learningand memory has been shown to be related to the generation of new neurons in the adult DG (Van et al., 2002;Nilsson et al., 1999).

It has been postulated only learning tasks which are hippocampal dependant affect progenitor cellproliferation and neurogenesis in the DG (Gould et al., 1999a). This idea has since been demonstrated using alearning task that is easily manipulated to be either hippocampal dependent or independent. Hippocampal-dependent learning can be assessed using trace eye blink conditioning. In trace conditioning the conditionedstimulus (CS), a tone, sounds for 5 seconds, then after a 100-1000 ms interval, the unconditioned stimulus(US) an airpuff or eyelid shock is activated. In this way the CS and US do not overlap. Hippocampal-independent learning can be assessed using delay eyeblink conditioning. In delay eyeblink conditioning thetone (CS) sounds for 5 seconds and in the last 20 ms of the tone sounding the airpuff (US) is activated. In thisway the CS and US overlap. Shors and colleagues used both trace and delay eyeblink conditioning todemonstrate that trace eyeblink conditioning, a hippocampal dependent task, is affected by neurogenesiswhereas delay eyeblink conditioning is not. Mice were treated with methylazoxymethanol acetate (MAM), ananti-mitotic agent which wipes out the progenitor cell population in the DG and administered BrdU to birthdate the cells then performed either trace or delay eyeblink conditioning. In both trace and delay eyeblinkconditioning, saline treated mice performed well on the task and had similar numbers of BrdU positive cells inthe DG. This is in contrast to mice treated with MAM which produced different results for trace and delayeyeblink conditioning. In trace eyeblink conditioning MAM severely impaired learning and obliterated BrdUincorporation in the DG, whereas, no impairment in learning was observed after delay eyeblink conditioningdespite mice being treated with MAM, thus obliterating the progenitor pool and resulted in a dramaticreduction of BrdU positive cells in the DG (Shors et al., 2001).These results clearly indicate that newlygenerated neurons in the adult DG are affected by the formation of hippocampal-dependent memory.

Only certain types of hippocampal dependent tasks have been shown to be involved in hippocampalneurogenesis (Shors et al., 2002). This was demonstrated using two different learning paradigms known torequire the HPC, the spatial navigation task and trace fear conditioning. Similar to the study mentionedearlier, mice were treated with MAM and BrdU then performance on either behavioral task was assessed. Thespatial navigation task is performed in the MWM and required the mouse to use spatial cues in theenvironment (like a black square on a wall) to navigate to and find the platform. Over trials mice learnedwhere the platform was located and spent less time trying to find it. MAM failed to result in impairment inescape latency but did significantly decreased BrdU+ cells in the SGZ, demonstrating that hippocampalprogenitor cell proliferation is not essential for this hippocampal-dependent task (Shors et al., 2002). In aseparate group of mice trace fear conditioning, which like trace eyeblink conditioning involves a time gapbetween CS and US presentation was performed. In trace fear conditioning MAM severely impaired learningand significantly diminished BrdU incorporation in the DG, thus providing more evidence for the involvementof trace conditioning in hippocampal neurogenesis (Shors et al., 2002). The above experiments clearlydemonstrate that some forms of learning are dependent on the HPC but not all hippocampal-dependentlearning tasks require neurogenesis.

The HPC is involved in the formation and expression of memory in the passive avoidance task in rats (Cahill &McGaugh, 1998). The logic underlying the passive avoidance (PA) task is that animals associate a particularenvironment with an unpleasant foot shock and learn by avoiding the environment they can avoid the aversivefoot shock. Consequently, an increase in response latency is thought to reflect the strength of the memory forthe aversive event (Sahgal & Mason, 1985). Specifically, the multi-herbal formula BR003 increased responselatency, and hence the memory of the foot shock while also increasing the number of BrdU positive cells in theDG (Oh et al., 2006). The PA task is relatively quick and simple but is limited in the information it providesregarding memory, that latencies increase following shock. A modified version of PA, the active avoidanceparadigm measures acquisition (learning), retention (memory) and the extinction of the conditioned response.



Active avoidance is a fear-motivated associative avoidance task. In this task the mouse has to learn to predictthe occurrence of an aversive event (shock) based on the presentation of a specific stimulus (tone), in order toavoid the aversive event by moving to a different compartment. The measures recorded include number ofavoidances (the mouse crossing to the other compartment during the warning signal), number of non-responses (the mouse failing to cross to the other compartment during the trial), response latency (latency toavoid or escape), number of intertrial responses (i.e., crossing the barrier within the intertrial interval), andserve as an index of learning which allows memory to be assessed.

Many studies supported the role of the HPC in active avoidance learning. LTP via electrical stimulation to theperforant pathway is negatively correlated to learning in the shuttle box avoidance task, suggesting activeavoidance training lowered the threshold frequency to induce LTP in the DG (Ramirez & Carrer, 1989). Activeavoidance learning increased the length of the postsynaptic density in the molecular cell layer of the DG (Vanet al., 1992) and increased immunoreactivity for muscarinic receptors in the granular cell layer (Van der Zee &Luiten, 1999). Two-way Active avoidance also increased synthesis of BDNF (Ulloor & Datta, 2005) and cAMPresponse element binding (CREB) in the dorsal HPC (Saha & Datta, 2005). Rats that learned the active shockavoidance task (responders) had similar levels of Brdu positive and Ki67 positive cells in the DG as non-responders, suggesting ASA has no effect on hippocampal progenitor cell proliferation (Van der et al., 2005).

Active avoidance testing is commonly used following exposure to severe inescapable foot shock in the learnedhelplessness model of depression. Exposure to inescapable foot shock decreased progenitor cell proliferationin the DG and this effect is reversed by chronic treatment with fluoxetine (Malberg & Duman, 2003). Onetarget of antidepressant treatment is BDNF since antidepressants not only increase the expression of CREB inthe rat HPC (Nibuya et al., 1996) but also increase the expression of BDNF (Nibuya et al., 1995). BDNFproduced antidepressant like effects in the learned helplessness model of depression (Shirayama et al., 2002).

Assessing NeurogenesisThe systemic injection of thymidine, radioactively labeled with tritium was the first method developed to labeldividing cells (Messier et al., 1958). Once in the bloodstream, tritiated thymidine competes with endogenousthymidine in all cells in the S phase of cell division and is permanently incorporated into the DNA. Labeledthymidine has a short half-life in vivo and labels all cells in the process of cell division when the label isinjected. At a later time point, tissue sections are prepared and coated with a photo emulsion. The radiationfrom the labeled thymidine molecules blackens the photo emulsion, thus making visible the typical grains ofthymidine autoradiography. Utilizing the tritiated thymidine method (Sidman et al., 1959; Messier et al., 1958;Messier & Leblond, 1960) Joseph Altman was able to demonstrate that the SVZ and the DG of the HPCproduce new neurons throughout the lifespan (Altman, 1962; Altman & Das, 1965; Altman, 1969).

BrdU is a false base that competes with endogenous thymidine and becomes permanently incorporated in theDNA during the S phase of the cell cycle. BrdU is typically administered via injections of usually 50-250 mg/kgin a single bout or over several days depending on the experimental paradigm (Corotto et al., 1993). BrdU isadvantageous because it is a permanent marker so any cells that express BrdU can be directly related to thetime BrdU was administered thus providing the birth date of the cell. It is important to note BrdU can beincorporated into cells that are on the verge of dying when cell death related mechanisms trigger DNArepair,therefore proper controls need to be included such as immunohistochemical stains for apoptosis(caspase-3 or TUNEL) and proliferative markers (Ki67) to determine if a cell is truly proliferative.

The rate of proliferation can be differentiated from the rate of survival by manipulating time between BrdUinjection and sacrifice so proliferating cells can be determined by sacrificing animals 24 hours after a BrdUinjection. In this way BrdU has time to incorporate into the cell but the cell does not have time to differentiateinto a neuron, a process which takes a minimum of 72 hours. Survival can be determined by taking the brainsof animals days, weeks or even months after BrdU injection. The phenotypic fate of the cell is determined inthe survival condition by double labeling BrdU with another marker. Table 1 presents a summary of thecommon markers used to determine the phenotype of neural progenitors.



Marker Significance Reference

Ki67 Proliferation; late G1, S, G2 and M phasesnuclear (Scholzen & Gerdes, 2000)

Doublecortin (DCX)Immature neuron; microtubule-associated protein enriched inmigratory neuronal cells. Early neuronal marker with lineagedetermined and limited self-renewaldendritic

(Meyer et al., 2002)

III β-tubulin (Tuj1) Immature neuron; Tubulin proteinsoma and processes

(Uittenbogaard & Chiaramello,2002)

Calretinin (CRT) Immature neurons; calcium binding protein transiently (Brandt et al., 2003)

Neuronal nuclei (NeuN)Mature neurons; mostly in nuclei but can be detected incytoplasmnucleus

(Mullen et al., 1992)

Glial Fibrillary AcidicProtein (GFAP) Intermediate filament protein expressed in astrocytes. (Fuchs & Weber, 1994)

Table 1. Antibodies used to assess phenotypic fate of progenitors.

If the cell expresses Ki67 it is an early progenitor since Ki67 is a protein expressed the G1, S, G2 and M phasesof the cell cycle (Scholzen & Gerdes, 2000). Cells that express doublecortin (DCX), β-tubulin III (III β-tubulin,Tuj1), or calretinin (CRT) are immature neurons. DCX is a microtubule associated protein transientlyexpressed in immature neurons (Meyer et al., 2002), Tuj1 marks tubulin in microtubules (Uittenbogaard &Chiaramello, 2002) and CRT is a calcium binding protein transiently expressed in immature neurons and isexpressed in the developing neuron at a stage where DCX expression dissipates (Brandt et al., 2003). The bestand most widely used marker to identify mature neurons is neuronal nuclei (NeuN) (Mullen et al., 1992). Theexpression of NeuN is restricted to post-mitotic neurons and is predominately located in the nucleus ofneurons although it can occasionally be observed in the neurites. In order to convincingly demonstrateneurogenesis, cells are double labeled with BrdU plus NeuN, which clearly demonstrates that the cell wasborn around the time of BrdU injection and survived to differentiate into a neuron. Cell survival depends onmany factors including the ability of the cell to form dendrites, an axon, synthesize neurotransmitter, receptorsand establish functional connections with other cells. Cells that don't establish functional connections willmost likely die.

It is possible to assess neurogenesis using methods other than the BrdU and tritiated thymidine methods.Using immunohistochemical and immunofluorescent techniques, cells can be stained for markers of immatureneurons that are transient and only present in newly formed neurons. Brandt and colleagues (2003) elegantlydemonstrated this method by defining time periods of development in which cells express particular markersdouble-labeled with BrdU (Brandt et al., 2003). BrdU is still the only way to birth date cells so for establishingthe method it was essential to know the exact age of cells. The expression of CRT plus BrdU positive cells wasgreatest 1 to 2.5 weeks after BrdU injection and the number of double-labeled cells was negligible at 4 weeks,demonstrating CRT is transient. If a cellexpressed DCX or CRT, that cell can be positively identified as animmature neuron, thus estimates of DCX or CRT positive cells in the DG represent estimates of neurogenesis.

Serotonergic Innervation in the Dentate GyrusSerotonin (5-HT) is a modulatory neurotransmitter in the central nervous system which is important in theregulation of vital brain functions such as feeding (Lucki, 1992), thermoregulation (Feldberg & Myers, 1964),sleep (Jouvet, 1967) and aggression (Sheard, 1969). In psychopathological states such as depression (Pinder &Wieringa, 1993), eating disorders (Leibowitz & Shor-Posner, 1986) and anxiety serotonergic signaling isdisturbed.

In the mammalian brain 5-HT is produced by neurons in the raphe nucleus (RN) which project to many areasof the brain via the medial forebrain bundle (MFB) (Azmitia & Segal, 1978; Parent et al., 1981). Neurons fromRN innervate virtually all brain areas with dense innervation occurring in the HPC, cerebral cortex, striatum,hypothalamus, thalamus, septum and olfactory bulb (Jacobs & Azmitia, 1992; Leger et al., 2001). Theinnervation of serotonergic fibers to areas within the HPC is variable (Moore & Halaris, 1975; Vertes et al.,1999; Bjarkam et al., 2003). The DG is innervated with serotonergic fibers in both the molecular layer and the



hilus with particularly dense innervation projecting to the SGZ where they synapse with interneurons (Halasy& Somogyi, 1993).

5-HT activates fifteen known receptors, many of which are expressed in the DG (El et al., 1989; Tecott et al.,1993; Vilaro et al., 1996; Djavadian et al., 1999; Clemett et al., 2000; Kinsey et al., 2001). Most of the 5-HTreceptors interact with G proteins except for the 5-HT3A receptors, which are ligand-gated ion channelreceptors. The 5-HT3 receptors (subtypes 5-HT3A and 5-HT3B) are ligand-gated Na+ ion channels and theiractivation leads to the depolarization of neurons (Barnes & Sharp, 1999). The 5-HT1 family of receptors(including subtypes 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E and 5-HT1F) are coupled to the Gi protein which, whenactivated decreases the activity of adenylyl cyclase thus decreasing the rate of formation of cyclic adenosinemonophosphate (cAMP). Activation of 5-HT1 receptors can lead indirectly to the opening of K+ channelstherefore increasing the conductance of the cell membrane for K+ ions. Activation of 5-HT4, 5-HT6, 5-HT7,receptors are coupled to Gs proteins which have the opposite effect. They increase the activity of adenylylcyclase, increase the rate of cAMP formation and decrease K+ conductance (Thomas et al., 2000; Raymond etal., 2001). The 5-HT2 receptors (including subtypes 5-HT2A, 5-HT2B, 5-HT2C) are coupled to Gq proteins andactivate phospholipase C (PLC), increasing the rate of formation of inositol triphosphate (IP3) anddiacylglycerol leading to the increased formation of protein kinase C (PKC) (Kurrasch-Orbaugh et al., 2003;Ananth et al., 1987).

Serotonin and Neurogenesis in the Dentate GyrusWhile several factors regulate the rate of generation of new cells in the adult DG, one of the most importantknown factors is 5-HT. Malberg and colleagues found increased levels of 5-HT resulted in the increased rate ofproliferation of neural progenitors in the DG (Malberg et al., 2000). Administering 5,7-dihydroxytryptamine(5,7-DHT), a serotonergic neurotoxin into the RN and caused the destruction of axons and serotonergic cellsand resulted in a decreased in the number of BrdU-labeled cells in the DG (Brezun & Daszuta, 1999). The 5,7-DHT lesion resulted in around a 60% depletion of the serotonergic innervation to the DG which lasted for onemonth. After two months, reinnervation to the DG was observed with the sprouting of serotonergic axons sothat by the third month there was no observable difference between the 5,7-DHT and vehicle in newlygenerated cells or serotonergic innervation (Brezun & Daszuta, 2000).

Many serotonergic receptors have been implicated in the regulation of neurogenesis in the DG. In vitro, whenthe 5-HT1A receptor agonist, 8-OH-DPAT was added to a medium in which cultured fibroblasts transfected withthe 5-HT1A receptor were present, the rate of cell divisions increased (Varrault et al., 1992). In vivo, 5-HT1A

receptor antagonists (NAN-190, p-MPPI and WAY-100635) decreased the number of progenitors in the DG byapproximately 30% (Radley & Jacobs, 2002) and injections of 5-HT1A receptor agonists increased the number ofBrdU positive cells in the DG (Santarelli et al., 2003). Similarly, Banasr and colleagues showed various 5-HT1

receptor agonists increase the number of BrdU labeled cells in the subgranular layer. Acute administration ofthe 5-HT2A/C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI), 5-HT2C receptor agonist RO 600175, and5-HT2C receptor antagonist SB 206553 had no effect of cell proliferation in the HPC, whereas the 5-HT2A/C

receptor antagonist ketanserin produced a 63% decrease in BrdU incorporation (Banasr et al., 2004). A recentstudy found acute ketanserin decreased proliferation whereas chronic ketanserin increased proliferation in theDG (Jha et al., 2008). No effect on proliferation in the DG was observed after DOI or lysergic acid diethylamide(LSD) were administered either acutely or once daily for seven consecutive days (chronic) (Jha et al., 2008).

The 5-HT2A receptor is involved in the regulation of BDNF in the HPC (Vaidya et al., 1997). DOI alone and incombination with selective 5-HT2A and 5-HT2C receptor antagonists decreased the expression of BDNF mRNA inthe HPC. Interestingly, the decrease in BDNF mRNA expression was blocked by the 5-HT2A receptor antagonistbut not the 5-HT2C receptor antagonist, implicating the 5-HT2A receptor in the regulation of BDNF expression.In addition, the stressinduced reduction in BDNF expression in the HPC was blocked by a 5-HT2A/C receptorantagonist (Vaidya et al., 1997).



Psilocybin (PSOP)PSOP (4-phosphoryloxy-N,N-dimethyltryptamine) is the main active agent in "magic mushrooms" and iscategorized as a indole hallucinogen. First isolated from Psilocybe mexicana, a mushroom from CentralAmerica by Albert Hofmann in 1957, PSOP was then produced synthetically in 1958 (Hofmann et al., 1958a;Hofmann et al., 1958b). PSOP is converted into the active metabolite psilocin (4-hydroxy-N,N-dimethyltryptamine) which may produce some of the psychoactive effects of PSOP. The chemical structure ofPSOP (C12H17N2O4P) and the metabolite, psilocin (C12H16N2O) are similar to 5-HT (C10H12N2O), the mainneurotransmitter which they affect (see Figure 1.3).

Figure 1.3. Chemical structure of Psilocybin, Psilocin and Serotonin.

In vivo studies in mice have shown the LD50 of PSOP via intravenous administration to be 280 mg/kg (Cerletti& Konzett, 1956; Cerletti, 1959). Autonomic effects of 10 mg/kg/sc in mice, rats, rabbits, cats and dogs includemydriasis, piloerection, irregularities in heart and breathing rate and hyperglycemic and hypertonic effects(Cerletti & Konzett, 1956; Cerletti, 1959). These effects were interpreted as an excitatory syndrome caused bystimulation of the sympathetic nervous system with one large exception being the absence of hyperlocomotion(Monnier, 1959).

PSOP exerts psychoactive effects by altering serotonergic neurotransmission by binding to 5-HT1A, 5-HT1D, 5-HT2A and 5-HT2C receptor subtypes (Passie et al., 2002). PSOP binds to the 5-HT2A receptor (Ki = 6 nM) withhigh affinity and to a much lesser extent to the 5-HT1A receptor subtype (Ki = 190 nM) (McKenna et al., 1990).However, PSOP has a lower affinity for 5-HT2A and 5-HT2C receptors compared to lysergic acid diethylamide(LSD), a similar indole hallucinogen (Nichols, 2004). In contrast to LSD, PSOP has a very low affinity to DAreceptors and only extremely high doses affect NE receptors. PSOP has been shown to induce schizophrenia-like psychosis in humans, a phenomenon attributed to the action of PSOP through 5-HT2A receptor action.Specifically, human volunteers were pretreated with ketanserin, an antagonist to the 5-HT2A/C receptor, thenadministered 0.25 mg/kg p.o. PSOP and the psychotomimetic effects of PSOP were completely blocked(Vollenweider et al., 1998). Since blocking the 5-HT2A receptor prevented the psychotropic effect of PSOP itappears as though the actions of PSOP are mediated via the activation of 5-HT2A receptors. A recent studytested PSOP-induced stimulus control and found 5-HT2A receptor antagonists prevented rats from recognizingPSOP in a drug discrimination task, an effect which was not observed with 5-HT1A receptor antagonists (Winteret al., 2007). Repeated daily administration of PSOP selectively downregulated 5-HT2A receptors in the ratbrain (Buckholtz et al., 1990; Buckholtz et al., 1988; Buckholtz et al., 1985). PSOP binds to the 5-HT2A receptorand stimulates arachidonic acid and consequently, the PI pathway resulting in the activation of PKC (Kurrasch-Orbaugh et al., 2003). This dissertation sought to evaluate the involvement of the 5-HT2A receptor in theregulation of hippocampal neurogenesis and hippocampal-dependent learning.

Specific AimsThe present study investigated the role of 5-HT2A receptor on hippocampal neurogenesis and hippocampal-dependent learning. The effects of acute and chronic 5-HT2A receptor agonists and an antagonist on thesurvival and phenotypic fate of progenitor cells in the DG were assessed using immunofluroescent techniques.



In addition the effects of acute PSOP on trace fear conditioning were used to assess learning and memory.

Specific Aim 1

To evaluate the effect of acute 5-HT2A receptor agonists and an antagonist on the survival and phenotypic fateof hippocampal progenitor cells. It was hypothesized PSOP and 251-NBMeO, both 5-HT2A receptor agonistspositively regulate neurogenesis in the DG of the HPC, and ketanserin, a 5-HT2A/C receptor antagonistdownregulates hippocampal neurogenesis.

Specific Aim 2

To elucidate whether PSOP affects learning and memory using the trace fear conditioning paradigm. It washypothesized acute exposure to PSOP would enhance hippocampal-dependent learning and ketanserin wouldimpair learning on the trace fear conditioning task.

Chapter TwoInvolvement of the 5HT2A receptor in the Regulation of

Adult Neurogenesis in the Mouse Hippocampus

AbstractSelective serotonin uptake inhibitors (SSRIs) are known to stimulate the production of new neurons in thehippocampus (HPC) by increasing synaptic concentration of serotonin (5-HT). The delay in the appearance ofantidepressant effects corresponds to the time required to generate new neurons. However, it is not clearwhich of the many serotonergic receptors in the HPC are responsible for the enhanced neurogenesis. Thecurrent study evaluated the effects of the acute and chronic administration of 5-HT2A agonists psilocybin and251-NBMeO and the 5-HT2A/C antagonist ketanserin on hippocampal neurogenesis. To investigate the effects ofacute drug administration mice received a single injection of varying doses of psilocybin, 251-NBMeO,ketanserin or saline followed by i.p. injections of 75 mg/kg bromodeoxyuridine (BrdU) for 4 consecutive daysfollowed by euthanasia two weeks later. For chronic administration 4 injections of psilocybin, ketanserin orsaline were administered weekly over the course of one month. On days following drug injections micereceived an injection of 75 mg/kg BrdU and were euthanized two weeks after the last drug injection. Unbiasedestimates of BrdU+ and BrdU/NeuN+ cells in the dentate gyrus (DG) revealed a significant dose dependentreduction in the level of neurogenesis after acute 5-HT2A receptor agonist or antagonist administration.Interestingly, chronic administration of psilocybin increased the number of newborn neurons in the DG whilethe antagonist suppressed hippocampal neurogenesis, suggesting the 5-HT2A receptor appears to be involvedin the regulation of hippocampal neurogenesis.

IntroductionEvidence suggests neurogenesis occurs throughout the lifespan in two specific regions of the adult brain, thesubventricular zone (SVZ) and the subgranular zone (SGZ) of the DG (Altman J, 1962; Altman & Das, 1965;Altman J, 1969). The proliferation and survival of neural progenitors in the adult HPC can be influenced by avariety of stimuli including stress, age, physical activity and depression (Gould et al., 1992; Kempermann etal., 1998; Van et al., 1999b; Malberg et al., 2000). Antidepressant medications such as selective 5-HT uptakeinhibitors (SSRIs) enhance the production of new born neurons in the DG of the HPC (Malberg et al., 2000).However, this effect is time specific with chronic administration (14 days or more) enhancing neurogenesis butnot acute treatment (Malberg et al., 2000). Interestingly, there is a delay in the appearance of antidepressanteffects which corresponds to the time required to generate new neurons (Santarelli et al., 2003) suggesting anenhancement of neurogenesis may mediate the behavioral effects of antidepressants.

The requirement of chronic administration of antidepressant medications to enhance neurogenesis is likely



due to number of factors. Antidepressant treatments upregulated the expression of brain-derived neurotrophicfactor (BDNF) in the HPC (Nibuya et al., 1995). BDNF knockout mice have diminished levels of neurogenesisin the DG (Lee et al., 2002) and infusions of BDNF into the lateral ventricles induced neurogenesis originatingin the SVZ (Pencea et al., 2001).

The involvement of 5-HT in the regulation of neurogenesis may be mediated through different 5-HT receptorsubtypes expressed on cells in the neurogenic microniche (Barnes & Sharp, 1999). The 5-HT2A receptor isinvolved in the regulation of BDNF in the HPC (Vaidya et al., 1997). 2,5-dimethoxy-4- iodoamphetamine (DOI),a 5-HT2A/C receptor agonist decreased the expression of BDNF mRNA in the HPC (Vaidya et al., 1997).Interestingly, the decrease in BDNF mRNA expression was blocked by the 5-HT2A receptor antagonist but notthe 5-HT2C receptor antagonist, implicating the 5-HT2A receptor in the regulation of BDNF expression in theHPC (Vaidya et al., 1997). Acute administration of DOI, 5-HT2C receptor agonist RO 600175, or the 5-HT2C

receptor antagonist SB 206553 had no effect on cell proliferation, whereas the 5-HT2A/C receptor antagonistketanserin produced a 63% decrease in BrdU incorporation (Banasr et al., 2004). A recent study found acuteketanserin decreased proliferation whereas chronic ketanserin increased proliferation in the DG (Jha et al.,2008). No effect on proliferation in the DG was observed after DOI or lysergic acid diethylamide (LSD) wereadministered either acutely or once daily for seven consecutive days (chronic) (Jha et al., 2008). However,daily doses of LSD or psilocybin (PSOP) produce rapid tolerance to the drug and resulted in a selectivedownregulation of the 5-HT2A receptor (Buckholtz et al., 1990; Buckholtz et al., 1985). Therefore, in order toinvestigate the role of the 5-HT2A receptor in the regulation of hippocampal neurogenesis the current studyevaluated the effects of acute and repeated intermittent administration of 5-HT2A agonists and the 5-HT2A/C

antagonist ketanserin on hippocampal neurogenesis.

Materials and Methods

Subjects

C57BL/6J male mice (30-40g) were housed in standard laboratory cages and left undisturbed for 1 week afterarrival at the animal facility. All mice had unlimited access to water and laboratory chow and were maintainedin a temperature and humidity controlled room on a 12:12 light/dark cycle with light onset at 7:00 AM. AllNational Institutes for Health (NIH) guidelines for the Care and Use of Laboratory Animals were followed(National Institutes of Health, 2002).

Drugs

251-NBMeO was synthesized in the laboratory of Dr David Nichols (Braden et al., 2006). PSOP was providedby Dr Francisco Moreno from University of Arizona. Ketanserin (+)-tartrate salt (#S006, St. Louis, MO) and 5-Bromo-2′-deoxyuridine (#B5002, St. Louis, MO) were supplied by Sigma-Aldrich Inc.

General Procedure

Acute Administration: A total of 48 C57BL/6 mice received a single injection of 0.1 mg/kg PSOP (n=6), 0.5mg/kg PSOP (n=6), 1.0 mg/kg PSOP (n=6), 0.1 mg/kg 251-NBMeO (n=6), 0.3 mg/kg 251-NBMeO (n=6), 1.0mg/kg 251-NBMeO (n=6), 1.0 mg/kg ketanserin (n=6) or saline (n=6). Mice received an intraperitoneal (i.p.)injection of 75 mg/kg BrdU once daily for 4 days following drug administration and were euthanized two weeksafter the last drug injection. Mice were euthanized with nembutal then transcardially perfused with 0.9%saline followed by 4% paraformaldehyde. Brains were stored in 4% paraformaldehyde, transferred to 20%sucrose solution and sectioned coronally using a cryostat (Leica, Germany) at 30µM in a 1:6 series and storedin 24-well plates in cryoprotectant at -20°C. Repeated Intermittent Administration: A total of 31 C57BL/6 micereceived 4 i.p. injections of either 0.5 mg/kg PSOP (n=6), 1.0 mg/kg PSOP (n=7), 1.5 mg/kg PSOP (n=6), 1.0mg/kg ketanserin (n=6) or 0.9% saline solution (n=6) over the course of one month on days 1, 8, 15, and 22.Each day following drug administration 75 mg/kg BrdU was injected i.p. All mice were euthanized two weeksafter the last drug injection according to the above procedures.



Immunofluorescence

For the double labeling of progenitor cells in the DG free-floating sections were denatured using 2N HCl andneutralized in 0.15M borate buffer then washed in PBS. Tissue was blocked in PBS+ (PBS, 10% normal goatserum, 1% 100x Triton X, 10% BSA) for 1 hour at 4°C and incubated for 48 hours at 4°C in an antibodycocktail of rat monoclonal anti-BrdU (AbD Serotec, Raleigh NC, #OBT0030G, 1:100) plus mouse anti-NeuN(Chemicon, 1:100) in PBS. Sections were washed in PBS and incubated in a secondary antibody cocktail ofgoat anti-rat IgG Alexa Fluor 594 (1:1000, Invitrogen, Eugene OR) plus goat anti-mouse (1:400, Invitrogen)and coated with vectorshield mounting medium (Invitrogen).

Quantitation

For the quantification of doubled labeled cells using immunofluroescence, the number of BrdU+ andBrdU/NeuN+ labeled cells were estimated using every 6th section taken throughout the DG (every 180microns). To avoid counting partial cells a modification to the optical dissector method was used so that cellson the upper and lower planes were not counted. The number of BrdU+ cells counted in every 6th section wasmultiplied by 6 to get the total number of BrdU+ or BrdU/NeuN+ cells in the DG (Shors et al. 2002). Positivelabeling was verified by confocal microscopy (Zeiss).

Design and analyses

Separate one-way analyses of variance (ANOVA) were used to evaluate the acute and chronic effects of 5-HT2A

receptor agonists and an antagonist on hippocampal neurogenesis. For acute drug administration separateone-way ANOVA was used to determine the effects of Drug [PSOP (saline, 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg),251-NBMeO (saline, 0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg)] and a two-tailed t-test (saline, 1.0 mg/kg ketanserin)was used to establish differences in cell survival and neurogenesis. For chronic drug administration a separateone-way ANOVA was used to determine the effects of Dose (saline, 0.5 mg/kg PSOP, 1.0 mg/kg PSOP, 1.5mg/kg PSOP, 1.0 mg/kg ketanserin) on cell survival and neurogenesis. When appropriate, post hoc analysessuch as Bonferroni were used to isolate Drug effects. All statistical analyses were determined significant at the0.05 alpha level.

Results

Effects of Acute Administration of 5-HT2A receptor agonists and anantagonist in vivo on Cell Survival and Neurogenesis in the Hippocampus

In order to investigate the effects of acute PSOP administration on cell survival and neurogenesis mice (n =6-7 per condition) were injected with PSOP (0.1, 0.5, or 1.0 mg/kg), 251-NBMeO (0.1 mg/kg, 0.3 mg/kg, 1.0mg/kg), ketanserin (1.0 mg/kg) or 0.9% saline solution then received 75 mg/kg BrdU once daily for 4 daysfollowing drug administration followed by euthanasia two weeks after the last drug injection. A one-wayANOVA detected significant differences in the total number of surviving BrdU+ cells in the DG as a result ofacute PSOP treatment [F(3,20)=6.64, p=0.003]. As can be seen in Figure 2.1A a significant decrease in thenumber of surviving BrdU+ cells was observed after 1.0 mg/kg PSOP compared to saline (indicated by *).

The phenotypic fate of surviving cells was determined by immunofluorescent labeling of BrdU and NeuN. Aone way ANOVA revealed a significant effect of dose on the number of double labeled neurons in the DG[F(3,20)=10.26, p=0.0003]. As can be seen in Figure 2.1B, acute administration of 1.0 mg/kg PSOPsignificantly diminished the number of BrdU/NeuN+ cells compared to saline (p<0.05). These data suggestacute administration of 1.0 mg/kg PSOP, a 5-HT2A agonist downregulated neurogenesis in the DG.

A one-way ANOVA detected significant differences in the total number of surviving BrdU+ cells in the DG as aresult of acute 251-NBMeO treatment [F(3,20)=9.00, p=0.0004]. There was a significant decrease in thenumber of surviving BrdU+ cells after acute administration of 0.1, 0.3 and 1.0 mg/kg 251- NBMeO comparedto saline (see figure 2.3A, indicated by *).



Figure 2.1. Effect of Acute PSOP Administration on Hippocampal Neurogenesis. Mice (n = 6 per condition) were injectedwith PSOP (0.1, 0.5, or 1.0 mg/kg) or saline then received 75 mg/kg BrdU once daily for 4 days following drugadministration followed by euthanasia two weeks after drug injection. A) The total number of BrdU+ cells in the DG weresignificant diminished after a single injection of 1.0 mg/kg PSOP (p<0.05). B) Acute administration of 1.0 mg/kg PSOPsignificantly diminished the number of BrdU/NeuN+ cells compared to saline (p<0.05). These data suggest acuteadministration of 1.0 mg/kg PSOP, a 5-HT2A agonist downregulated neurogenesis in the DG of the HPC.* indicates a significant difference from saline.

Figure 2.2. Representative photomicrographs showing the effects of acute PSOP on hippocampal neurogenesis. NeuN+cells (left), BrdU+ cells (center) and NeuN/BrdU+ cells (right). Saline (A-C), 0.1 mg/kg PSOP (D-F), 0.5 mg/kg PSOP (G-I)and 1.0 mg/kg PSOP (J-L). Scale = 50 µM.



Figure 2.3. Effect of the selective 5-HT2A receptor agonist, 251-NBMeO on hippocampal neurogenesis. Mice (n = 6 percondition) were injected with 251- NBMeO (0.1, 0.3, or 1.0 mg/kg) or saline then received 75 mg/kg BrdU once daily for 4days following drug administration followed by euthanasia two weeks after drug injection. A) There was a significantdecrease in the total number of BrdU+ cells in the DG after the administration of 251-NBMeO (p<0.05). B) The number ofnew born neurons was significantly decreased after 1.0 mg/kg of 251- NBMeO compared to saline (p<0.05), suggestingacute administration of the 5-HT2A receptor agonist downregulated neurogenesis in the DG of the HPC.* indicates a significant difference from saline.

Figure 2.4. Representative photomicrographs showing the effects of the selective 5-HT2A receptor agonist 251-NBMeOon hippocampal neurogenesis. NeuN+ cells (left), BrdU+ cells (center) and NeuN/BrdU+ cells (right). Saline (AC), 0.1mg/kg 251-NBMeO (D-F), 0.3 mg/kg 251-NBMeO (G-I) and 1.0 mg/kg 251-NBMeO (J-L). Scale = 100 µM.



In addition, there was a significant effect of drug on the number of double labeled neurons in the DG[F(3,20)=3.00, p=0.03]. As can be seen in Figure 2.3B, 1.0 mg/kg 251-NBMeO significantly diminished thenumber of new born neurons in the DG compared to saline (p<0.05, indicated by *). These data suggest acuteadministration of the selective 5-HT2A receptor agonist, 251- NBMeO, attenuated hippocampal neurogenesis

Interestingly, acute administration of the 5-HT2A/C receptor antagonist ketanserin produced similar effects onneurogenesis as high doses of the 5-HT2A receptor agonists. As can be seen in figure 2.5, the total number ofBrdU+ cells in the DG was significantly decreased after 1.0 mg/kg ketanserin [t(10)=3.0, p=0.008], suggestingthat the 5-HT2A/C receptor is involved in the regulation of cell survival in the HPC. Furthermore, acuteketanserin decreased the total number of BrdU/NeuN positive cells compared to saline [t(10)=3.0, p=0.02]demonstrating that antagonism of the 5-HT2A/C receptor negatively regulated the number of new born neuronsgenerated in the DG of the HPC.

Figure 2.5. Acute Administration of the 5-HT2A/C receptor antagonist ketanserin negatively regulated cell survival and neurogenesis inthe HPC. Mice (n = 6 per condition) were injected with 1.0 mg/kg ketanserin or saline then received 75 mg/kg BrdU once daily for 4days following drug followed by euthanasia two weeks after drug injection. Ketanserin decreased the total number of BrdU+ (A) andBrdU/NeuN positive cells (B) suggesting that antagonism of the 5-HT2A/C receptor negatively regulated cell survival and neurogenesisin the HPC.

Figure 2.6. Representative photomicrographs showing the effects of the 5-HT2A/C receptor antagonist ketanserin on neurogenesis in thedentate gyrus. NeuN+ cells (left), BrdU+ cells (center) and NeuN/BrdU+ cells (right). Saline (A-C), 1.0 mg/kg ketanserin (D-F). Scale =100 µM.



Effects of Repeated Intermittent PSOP Administration on Progenitor CellSurvival and Neurogenesis in the Hippocampus

In order to investigate the effects of repeated intermittent PSOP administration on cell survival mice (n = 6- 7per condition) were injected with PSOP (0.5, 1.0, or 1.5 mg/kg), 1.0 mg/kg ketanserin or 0.9% saline solutiononce weekly for 4 weeks. Each day following drug administration, mice were administered 75 mg/kg BrdU andsacrificed two weeks after last drug injection. ANOVA failed to reveal differences in the total number of BrdU+cells in the DG as a result of repeated intermittent drug treatment [F(4,26)=2.20, p=0.09]. As can be seen inFigure 2.7A a trend toward an increase in the number of surviving cells was observed after high doses ofPSOP.

Figure 2.7. Effect of Repeated Intermittent PSOP Administration on Hippocampal Neurogenesis. Mice (n = 6-7 per condition) wereinjected with PSOP (0.5, 1.0, or 1.5 mg/kg), 1.0 mg/kg ketanserin or saline once weekly for 4 weeks. Each day following drugadministration, mice were administered 75 mg/kg BrdU and sacrificed two weeks after last drug injection. A) The total number ofBrdU+ cells in the DG did not differ as a result of repeated intermittent drug treatment, however, a trend toward an increase in thenumber of surviving cells was observed after high doses of PSOP. B) Repeated intermittent administration of 1.5 mg/kg PSOPsignificantly increased the number of BrdU/NeuN+ cells compared to saline and ketanserin (p<0.05). These data suggest repeatedintermittent administration of high doses of PSOP, a 5-HT2A agonist upregulated neurogenesis in the DG.* indicates a significant difference from saline and ketanserin.

The phenotypic fate of surviving cells was determined by immunofluorescent labeling of BrdU and NeuN.ANOVA revealed a significant effect of Dose on the number of double labeled neurons in the DG [F(4,26)=3.15,p=0.02]. As can be seen in Figure 2.7B, chronic administration of 1.5 mg/kg PSOP significantly increased thenumber of BrdU/NeuN+ cells compared to saline and ketanserin (p<0.05) (indicated by *). These data suggestrepeated intermittent administration of PSOP, a 5-HT2A agonist upregulated neurogenesis in the DG of theHPC.



Figure 2.8. Representative photomicrographs showing the effects of repeated intermittent PSOP or ketanserin administration onneurogenesis in the DG. NeuN+ cells (left), BrdU+ cells (center) and NeuN/BrdU+ cells (right). Saline (AC), 0.5 mg/kg PSOP (D-F), 1.0mg/kg PSOP (G-I), 1.5 mg/kg PSOP (J-L), and 1.0 mg/kg Ketanserin (M-O). Scale = 100 µM.



DiscussionThe present investigation illustrates the involvement of the 5-HT2A receptor in the regulation of neurogenesisin the DG of the HPC. Acute administration of low doses of PSOP (0.1 and 0.5 mg/kg) did not alterneurogenesis, however, higher doses of PSOP (1.0 mg/kg) decreased neurogenesis two weeks after drugexposure (Figure 2.1). In addition, acute administration of the potent 5-HT2A receptor agonist 251-NBMeO(Figure 2.3) and the 5-HT2A/C receptor antagonist ketanserin (Figure 2.5) decreased hippocampal neurogenesis.Acute ketanserin (1-5 mg/kg) administered within 4 hours of sacrifice decreased the number of BrdU+ cells inthe DG, indicating a reduction in cell proliferation (Banasr et al., 2004; Jha et al., 2008). The present studyextends these findings by demonstrating that acute ketanserin decreases the number of BrdU+ andBrdU/NeuN+ cells 2 weeks after drug administration, indicating a reduction in cell survival and neurogenesisafter exposure to acute ketanserin.

The current study reports that repeated intermittent administration of high doses of PSOP (1.5 mg/kg)increased neurogenesis in the DG (see Figure 2.7). A recent study investigated the effects of chronic DOI, LSDand ketanserin administration on the number of BrdU+ cells in the DG (Jha et al., 2008). They report no effectof chronic DOI or LSD on progenitor cell proliferation but observed an increase in the number of BrdU+ cellsin the DG after chronic ketanserin. There are several methodological differences between the studies whichmay account for the different results, namely drug administration protocol, doses of compounds administeredand administration protocol of BrdU. Jha and colleagues administered DOI (8 mg/kg), LSD (0.5 mg/kg) orketanserin (5 mg/kg) once daily for seven consecutive days for the chronic drug administration protocol (Jha etal., 2008). The current investigation administered PSOP (0.5, 1.0 or 1.5 mg/kg) or ketanserin (1.0 mg/kg) 4times over the course of one month so that injections were given one week apart. This was a criticalconsideration in our experimental design given that daily doses of LSD, PSOP or other 5-HT2A receptoragonists produce rapid tolerance to the drug and results in a selective downregulation of the 5-HT2A receptor(Buckholtz et al., 1990; Buckholtz et al., 1985; Buckholtz et al., 1988). Jha and colleagues administered BrdU(200 mg/kg) 2 hours after the last injection of DOI, LSD or ketanserin and sacrificed the animals 24 hours later(Jha et al., 2008). In the current study BrdU (75 mg/kg) was administered 24 hours after each drug injectionand mice were sacrificed two weeks after the last drug injection so that time between the first BrdU injectionand sacrifice was 6 weeks. This allowed for the assessment of neurogenesis giving time for the birth-datedcells (BrdU labeled) to mature into neurons.

Brain derived neurotropic factor (BDNF) has been implicated in synaptic plasticity (Kang et al., 1997; Pang etal., 2004; Tyler et al., 2002) through the modulation of synapse formation and dendritic spine growth in theHPC (Bamji et al., 2006; Tyler & Pozzo-Miller, 2001; Tyler & Pozzo-Miller, 2003). Chronic administration of 5-HT agonists (including SSRIs) upregulate BDNF mRNA expression in the HPC (Nibuya et al., 1995; Nibuya etal., 1996). Evidence suggests that the 5-HT2A receptor is involved in the regulation of BDNF in the HPC (Vaidyaet al., 1997). Specifically DOI, a 5-HT2A/C receptor agonist decreased BDNF mRNA expression in the granulecell layer of the DG but not in the CA subfields of the HPC. Interestingly, the decrease in BDNF mRNAexpression was blocked by the 5-HT2A receptor antagonist but not the 5-HT2C receptor antagonist, implicatingthe 5-HT2A receptor in the regulation of BDNF expression (Vaidya et al., 1997).

PSOP and 251-NBMeO exert their effects through binding to 5-HT receptors. PSOP binds to the 5-HT2A

receptor (Ki = 6 nM) with high affinity and to a much lesser extent to the 5-HT1A receptor subtype (Ki = 190nM) (McKenna et al., 1990). The synthetic phenethylamine 251-NBMeO binds to 5-HT2A receptors (Ki = 0.044nM) with an extremely high affinity (Braden et al., 2006).

5-HT2A receptors are highly expressed throughout the HPC in the DG, hilus, CA1, and CA3 and are colocalizedon GABAergic neurons, pyramidal and granular cells (Cornea-Hebert et al., 1999; Morilak et al., 1993;Pompeiano et al., 1994; Shen & Andrade, 1998; Luttgen et al., 2004; Morilak et al., 1994). 5-HT2A receptoragonists stimulate arachidonic acid and consequently, the phosphoinositide (PI) pathway resulting in theactivation of protein kinase C (PKC) (Kurrasch-Orbaugh et al., 2003; Ananth et al., 1987). Electrophysiologicalevidence suggests that 5-HT2A receptors stimulate GABAergic interneurons in the HPC (Shen & Andrade,1998) and GABAergic interneurons in the hilus form connections with progenitor cells in the SGZ (Wang et al.,2005). When progenitor cells are less than 2 weeks old the GABAergic input exerts an excitatory influence onthe progenitor cells and as the cells establish glutamatergic synapses the GABAergic interneurons become



inhibitory (Wang et al., 2005; Zhao et al., 2006; Aimone et al., 2006). Given that 5-HT2A receptor agonistsadministered chronically downregulates receptor expression, and evidence suggests that the 5-HT2A receptorexcites the GABAergic interneurons which stimulate progenitor cells in the SGZ, one might anticipate areduction in neurogenesis after chronic PSOP. On the contrary, the present study reports high doses of PSOPupregulates neurogenesis. Based on this finding it is plausible to suggest the increase in neurogenesisobserved may be attributed to the administration paradigm in which PSOP was given 4 times over the courseof one month so that injections were given once a week. In this administration paradigm alterations inreceptor levels may not have occurred to the extent that occurs with daily exposure and give this highly plasticmicroniche time to adapt between drug exposures.

The results shown provide evidence that the 5-HT2A receptor is involved in the regulation of hippocampalneurogenesis. The data suggest that acute administration of 5-HT2A receptor agonists and an antagonistdownregulated neurogenesis in the DG. Whereas, chronic administration of high doses of 5-HT2A receptoragonists enhance hippocampal neurogenesis in the DG. Future studies should investigate the effects of chronicadministration of PSOP and ketanserin on 5-HT2A receptor levels and neuroplasticity in the HPC.

AcknowledgmentsThis work was supported by the Helen Ellis Research Endowment (JSR). Thanks to David Nichols Ph.D. fordonating the selective 5-HT2A agonist 251-NBMeO and Dr Francisco Moreno from University of Arizona andRick Doblin Ph.D. of the Multidisciplinary Association for Psychedelic Studies (MAPS) for donating the PSOP.

Chapter ThreeThe Effects of Psilocybin on Trace Fear Conditioning

AbstractAberrations in brain serotonin (5-HT) neurotransmission have been implicated in psychiatric disordersincluding anxiety, depression and deficits in learning and memory. Many of these disorders are treated withdrugs which promote the availability of 5-HT in the synapse. However, it is not clear which of the 5-HTreceptors are involved in behavioral improvements. The current study aimed to investigate the effects ofpsilocybin, a 5-HT2A receptor agonist on hippocampal-dependent learning. Mice received a single injection ofpsilocybin (0.1, 0.5, 1.0 or 1.5 mg/kg), ketanserin (a 5-HT2A/C antagonist) or saline 24 hours before habituationto the environment and subsequent training and testing on the fear conditioning task. Trace fear conditioningis a hippocampal-dependent task in which the presentation of the conditioned stimulus (CS, tone) is separatedin time by a trace interval to the unconditioned stimulus (US, shock). All mice developed contextual and cuedfear conditioning; however, mice treated with psilocybin extinguished the cued fear conditioning more rapidlythan saline treated mice. Interestingly, mice given the 5-HT2A/C receptor antagonist ketanserin showed less ofcued fear response than saline and psilocybin treated mice. Future studies should examine the temporaleffects of acute and chronic psilocybin administration on hippocampal-dependent learning tasks.

IntroductionThe hippocampus (HPC) plays a critical role in learning tasks that involve temporal encoding of stimuli (Squireet al., 1992; Squire, 1992). The trace classical conditioning paradigm requires temporal processing becausethe conditioned stimulus (CS) and the unconditioned stimulus (US) are separated in time by a trace interval.Lesions to the HPC prevent trace conditioning, indicating that it is a hippocampal-dependent task (McEchronet al., 1998; Weiss et al., 1999).

The serotonergic system has been implicated in hippocampal-dependent learning. Administration of selectiveserotonin (5-HT) uptake inhibitors (SSRIs) produce alterations in performance on learning tasks that requirethe HPC (Flood & Cherkin, 1987; Huang et al., 2004). In a knockout (KO) mouse model, central 5-HT deficientmice developed heightened contextual fear conditioning which was reversed by intracerebroventricular



microinjection of 5-HT (Dai et al., 2008). An impairment in learning on the morris water maze was observed in5-HT1A KO mice along with functional abnormalities in the HPC (Sarnyai et al., 2000). Activation of 5-HT1A

receptors in the medial septum alters encoding and consolidation in a hippocampal-dependent memory task(Koenig et al., 2008). In addition, Lysergic acid diethylamide (LSD), a 5-HT2A receptor agonist facilitatedlearning of a brightness discrimination reversal problem (King et al., 1972; King et al., 1974).

Evidence suggests that performance on hippocampal-dependent learning tasks is influenced by neurogenesisin the dentate gyrus (DG) of the HPC (Van et al., 2002; Nilsson et al., 1999; Shors et al., 2001; Shors et al.,2002; Gould et al., 1999a; Gould et al., 1999c). This was elegantly demonstrated by Shors and colleagues bytreating animals with methylazoxymethanol acetate (MAM), an anti-mitotic agent which eradicates theprogenitor cell population in the DG before testing mice on hippocampal-dependent and hippocampal-independent learning tasks (Shors et al., 2001; Shors et al., 2002). MAM treated animals had significantlyfewer BrdU+ cells in the subgranular zone (SGZ) of the DG but showed no impairment in the spatialnavigation task (HPC-dependent) or delay eyeblink conditioning task (HPC-independent) demonstrating thatthe hippocampal progenitor cell population is not essential for these particular tasks (Shors et al., 2002; Shorset al., 2001). In contrast, MAM severely impaired performance on trace fear conditioning and trace eyeblinkconditioning, providing evidence for the involvement of progenitor cells in the DG in trace classicalconditioning (Shors et al., 2002; Shors et al., 2001). In addition, hippocampal neurogenesis is influenced byserotonergic agonists. Specifically, SSRIs enhance the production of new born neurons in the DG of the HPC(Malberg et al., 2000; Santarelli et al., 2003).

Psilocybin (PSOP), a tryptamine alkaloid, exerts psychoactive effects by altering serotonergicneurotransmission (Passie et al., 2002). PSOP binds to the 5-HT2A receptor (Ki = 6 nM) with high affinity and toa much lesser extent to the 5-HT1A receptor subtype (Ki = 190 nM) (McKenna et al., 1990). 5-HT2A receptorsare highly expressed throughout the HPC in the DG, hilus, CA1, and CA3 (Cornea-Hebert et al., 1999; Morilaket al., 1993; Pompeiano et al., 1994; Shen & Andrade, 1998; Luttgen et al., 2004; Morilak et al., 1994). 5-HT2A

receptor agonists, including PSOP, stimulate arachidonic acid (AA) and consequently, the phosphoinositide (PI)pathway resulting in the activation of protein kinase C (PKC) (Kurrasch-Orbaugh et al., 2003; Ananth et al.,1987). Electrophysiological evidence suggests that 5-HT2A receptors stimulate GABAergic interneurons in theHPC (Shen & Andrade, 1998) and GABAergic interneurons in the hilus form connections with progenitor cellsin the SGZ (Wang et al., 2005).

The present study aimed to investigate the effects of the 5-HT2A receptor agonist PSOP on hippocampal-dependent learning. Mice received a single injection of psilocybin (0.1, 0.5, 1.0 or 1.5 mg/kg), 1.0 mg/kgketanserin (a 5-HT2A/C antagonist) or saline 24 hours before habituation to the environment and subsequenttraining and testing on the trace fear conditioning task.

Materials and Methods

Subjects

C57BL/6J male mice (30-40g) were housed in standard laboratory cages and left undisturbed for 1 week afterarrival at the animal facility. All mice had unlimited access to water and laboratory chow and were maintainedin a temperature and humidity controlled room on a 12:12 light/dark cycle with light onset at 7:00 AM. AllNational Institutes for Health (NIH) guidelines for the Care and Use of Laboratory Animals were followed(National Institutes of Health, 2002).

General Procedure

Mice (n=9-10/condition) received an intraperitoneal (i.p.) injection of PSOP (0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg,1.5 mg/kg), ketanserin (1.0 mg/kg) or 0.9% saline and 24 h later were habituated to the testing chamber for 30min. The fear conditioning environment consisted of two chambers each placed inside a larger soundproofchamber. The 35.6 (W) x 38.1 (D) x 31.8 (H) cm freeze monitor box (San Diego Instruments, San Diego, CA) isa clear Plexiglas chamber with a removable lid which contains a metal grid floor (0.3 cm grids spaced 0.8 cmapart) through which a foot shock can be delivered. Photobeam activity within the chamber recorded the



vertical and horizontal movements of mice. Two minutes into the habituation period a baseline (BL) measure ofmovement was recorded for 3 minutes and served as the habituation BL measure. Freeze monitor boxes werecleaned with quatricide between each mouse to prevent olfactory cues. Mice were returned to their home cageafter habituation.

The next day mice were returned to the same freeze monitor chamber and underwent training to form CS - USassociations. After a 2 minute acclimation period, mice were exposed to 10 trials of trace fear conditioningwhich is illustrated in Figure 3.1. Each trial consisted of the CS (tone, 82 dB, 15 s) followed by a trace interval(30 s) and ended with the presentation of the US (shock, 0.5 s, 1 mA) delivered through the grid flooring. Aftereach trial ended there was a 210 s intertrial interval (ITI). Freeze monitor boxes were cleaned with quatricidebetween each mouse to prevent olfactory cues and mice were returned to their home cage after training.

Figure 3.1. Schematic representation of the Trace Fear Conditioning Paradigm. Trace fear conditioning is a hippocampal-dependenttask in which the presentation of the conditioned stimulus (CS, tone) is separated in time by a trace interval to the unconditionedstimulus (US, shock).

On day 3 of the task, testing of the fear conditioning response was assessed in 2 phases. First, mice wereplaced in the freeze monitor box for 5 minutes and movement was recorded for the last 3 minutes and used toassess the measure of fear associated to the training context. Mice were then returned to the home cage for 1hour. Second, the context was altered by replacing the grid floors with black Plexiglas flooring and adding acotton ball with 1ml vanilla essence inside the sound attenuated chamber. Mice were placed inside the novelchamber and after 2 minutes, movements were recorded for the next 3 minutes. Next 10 trials with thepresentation of the CS only were delivered with an ITI of 240 s. Cue fear conditioning was measure by thepercent freezing during the CS (tone; 15s), during the trace interval (30s) and after the trace when the USwould have occurred. Conditioned fear was defined as an increase in percent immobility during the cue test.Percent immobility was calculated by dividing time spent immobile during stimuli (CS, trace or after trace) bythe length of time the stimuli lasted multiplied by 100.

Design and analyses

Separate two-way repeated measure analyses of variance (ANOVA) were used to evaluate the effect of Doseand Trial on each dependent variable in the trace fear conditioning task. Dependent measures recordedincluded percent freezing during CS, during trace, after trace, during habituation BL, during the context testand in response to the novel environment. When appropriate, post hoc analyses such as Bonforreoni were usedto isolate effects. All statistical analyses will be determined significant at the 0.05 alpha level.

Results

Acquisition

The acquisition of the freezing response is displayed in Figure 3.2 showing both the response to the CS (Figure3.2A) and during the trace (Figure 3.2B). Using percent immobility in response to the CS for the first 3 trials oftraining, ANOVA showed that regardless of drug treatment there was a significant improvement across trial[F(2, 108) = 40.0, p<0.0001]. Specifically, immobility in response to the CS increased from trial 1 to trial 3indicating the learned association between the stimuli during training (p<0.05). Additionally, ANOVA of thepercent immobility during the trace interval revealed a significant effect of trial [F(2, 108) = 20.0, p<0.0001].There was a striking increase in the amount of time spent immobile during the trace period from trial 1 to trial3 demonstrating that the association between the CS and US promoted immobility in anticipation of the shock.



Figure 3.2. Effects of Psilocybin on the Acquisition of Trace Fear Conditioning. Mice underwent training to form CS - US associationsby exposure to 10 trials of trace fear conditioning. Each trial consisted of the presentation of the CS (tone, 15-s) followed by a traceinterval (30-s) and ended with the US (shock, 0.5-s). A) Percent immobility during presentation of the 15-s CS during the first threetrials of CS - US pairing. B) Percent immobility during the 30-s trace interval during the first three trials of CS - US pairing.

Contextual Fear Conditioning

Contextual fear conditioning was assessed by comparing percent immobility in the freeze monitor box onhabituation day to percent immobility during the context test. There was no interaction between Dose andTrial [F(5,90) = 0.95, p=0.45] and no effect of Dose during the habituation BL or context test [F(5,90) = 1.15,p=0.34]. However, there was a significant effect of Trial [F(1,90) = 105.85, p<0.0001] indicating that micespent significantly more time freezing after the CS - US pairings during the context test compared tohabituation BL. Figure 3.3 illustrates percent immobility during exposure to the freeze monitor box during thehabituation test (A) and during the contextual fear conditioning test (B).

Figure 3.3. Contextual Fear Conditioning. Percent immobility expressed during exposure to the freeze monitor box during habituation(A) and during contextual fear conditioning (B). All mice expressed contextual fear conditioning as indicated by a significant increasein percent immobility during the context test (p<0.05).

Cue Fear Conditioning

Freezing responses (% immobility) during the CS only (tone) test are illustrated in Figure 3.4. There was asignificant effect of Trial [F(2,102) = 7.83, p<0.0007] with trials 2 and 3 eliciting significantly more freezing inresponse to the CS compared to trial 1 (Figure 3.4A). There was also a significant effect of Dose [F(5,51) =4.96, p<0.0009] revealing that control mice showed a greater fear response to the cue compared to 0.1 mg/kg



PSOP, 1.0 mg/kg PSOP, 1.5 mg/kg PSOP and 1.0 mg/kg ketanserin. ANOVA revealed a significant effect ofDose during the trace interval [F(5,51) = 2.41, p<0.05]. The fear associated with the trace interval during thefirst three trials on test day was reduced in mice treated with 1.0 mg/kg PSOP compared to saline and 1.5mg/kg PSOP (Figure 3.4B). Figure 3.4C illustrates the fear response after the trace interval which coincideswith the timing the US (shock) was delivered during the acquisition of the CS - US pairing phase. A two-wayrepeated measures ANOVA revealed a significant interaction between Dose and Trial [F(10,100) = 3.53,p<0.0005]. Interestingly, mice administered low doses of PSOP (0.1 and 0.5 mg/kg) froze more on trial 1compared to trial 2 and 3 suggesting they are more apt to adapt to the absence of the US so that the fearresponse is diminished as the US is extinguished. This pattern was reversed in mice treated with ketanserinwho increased fear responses from trials 1 to 3, indicating the robust memory for the US even in its absence.Taken together, these data suggest differential effects of PSOP on trace fear conditioning.

Figure 3.4. Effect of Acute PSOP on Cue Fear Conditioning. Cue fear conditioning was examined 24 hours after the training period inwhich mice were exposed to 10 trials of CS - US pairings. A) Percent immobility during presentation of the 15-s CS during the firstthree CS-only trials. B) Percent immobility during the 30-s trace interval during the first three trials. C) Percent immobility after thetrace interval which coincides with the timing the US (shock) was delivered during the acquisition of the CS - US pairing phase.

DiscussionThe present investigation illustrates the involvement of the 5-HT2A receptor in trace fear conditioning, ahippocampal-dependent learning task. During the acquisition of the freezing response there was a strikingincrease in the amount of time spent freezing during the presentation of the CS and during the trace periodfrom trial 1 to trial 3 (see Figure 3.2). These data demonstrate that the association between the CS and USpromoted freezing in anticipation of the shock, however, the acquisition of learning was not altered by acuteadministration of PSOP or ketanserin.

All conditions displayed similar locomotor activity levels in the freeze monitor box during the habituationbaseline exposure and during re-exposure to the same environment during the contextual fear conditioningtest. As expected mice froze substantially more during re-exposure to the same context after CS - USassociations were formed compared to the habituation baseline, indicating that all groups formed contextualfear conditioning (Figure 3.3). It is well known that contextual fear conditioning is a hippocampal-dependentlearning task (Kim & Fanselow, 1992; McNish et al., 1997; Hirsh, 1974; Esclassan et al., 2008; Frohardt et al.,1999). The serotonergic system has been implicated in performance on the contextual fear conditioning task(Dai et al., 2008). The present investigation found that the 5-HT2A receptor does not alter contextual fearconditioning since no differences were observed between controls and mice treated with PSOP or ketanserin.

The current study reports alterations in cue associated fear conditioning mediated by the 5-HT2A receptor.Independent of drug administered all mice developed cue-induced freezing during the presentation of the toneon the CSonly trial (see Figure 3.4). At the time coinciding with the expected US (shock) presentation lowdoses of PSOP (0.1 and 0.5 mg/kg) elicited a heightened freezing response on trial 1 compared to other trialssuggesting they are more apt to adapt to the absence of the US so that the fear response is diminished as theUS is extinguished. This pattern was reversed in mice treated with ketanserin who increased fear responsesfrom trials 1 to 3, indicating the robust memory for the US even in its absence.



Synaptic plasticity in the HPC is critical for the acquisition of learning and memory. Brain derived neurotropicfactor (BDNF) has been implicated in synaptic plasticity and memory processing (Kang et al., 1997; Pang etal., 2004; Tyler et al., 2002) through the modulation of synapse formation and dendritic spine growth in theHPC (Bamji et al., 2006; Tyler & Pozzo-Miller, 2001; Tyler & Pozzo-Miller, 2003). Chronic administration of 5-HT agonists (including SSRIs) upregulate BDNF mRNA expression in the HPC (Nibuya et al., 1995; Nibuya etal., 1996).

Evidence suggests that the 5-HT2A receptor is involved in the regulation of BDNF in the HPC (Vaidya et al.,1997). Specifically DOI, a 5-HT2A/C receptor agonist decreased BDNF mRNA expression in the granule cell layerof the DG but not in the CA subfields of the HPC. Interestingly, the decrease in BDNF mRNA expression wasblocked by the 5-HT2A receptor antagonist but not the 5-HT2C receptor antagonist, implicating the 5-HT2A

receptor in the regulation of BDNF expression (Vaidya et al., 1997).

5-HT2A receptors are highly expressed throughout the HPC in the DG, hilus, CA1, and CA3 and are colocalizedon GABAergic neurons, pyramidal and granular cells (Cornea-Hebert et al., 1999; Morilak et al., 1993;Pompeiano et al., 1994; Shen & Andrade, 1998; Luttgen et al., 2004; Morilak et al., 1994). Agonists to the 5-HT2A receptor stimulate AA and consequently, the PI pathway resulting in the activation of PKC (Kurrasch-Orbaugh et al., 2003; Ananth et al., 1987). Electrophysiological evidence suggests that 5-HT2A receptorsstimulate GABAergic interneurons in the HPC (Shen & Andrade, 1998). Aberrations in GABAergic function inthe HPC has been implicated in learning and memory due to the role of hippocampal GABA in temporospatialintegration (Wallenstein et al., 1998).

Taken together, the data reported in the present investigation implicate the 5-HT2A receptor in hippocampal-dependent learning. The present study reports that prior exposure to PSOP altered responsivity in a novelenvironment indicating an absence of a fear response, an effect not elicited by control mice. Furthermore, lowdoses of PSOP heightened cue elicited fear conditioning and antagonists to the 5-HT2A/C receptor diminishedfear conditioning to the cue. Results of this study raise the possibility that 5-HT2A receptor activity could leadalterations hippocampal-dependent learning and memory.

AcknowledgmentsThis work was supported by the Helen Ellis Research Endowment (JSR). Thanks to Dr Francisco Moreno fromUniversity of Arizona and Rick Doblin Ph.D. of the Multidisciplinary Association for Psychedelic Studies(MAPS) for donating the PSOP.

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